- Email: [email protected]
sorbitol co-feeding
Accepted Manuscript Title: Transcriptional analysis for carbon metabolism and kinetic modeling for heterologous proteins productions by Pichia pastoris in induction process with methanol/sorbitol co-feeding Authors: Luqiang Jia, Enock Mpofu, Tingyong Tu, Qiangqiang Huai, Jiaowen Sun, Shanshan Chen, Jian Ding, Zhongping Shi PII: DOI: Reference:
S1359-5113(17)30153-8 http://dx.doi.org/doi:10.1016/j.procbio.2017.05.011 PRBI 11040
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
23-1-2017 6-5-2017 10-5-2017
Please cite this article as: Jia Luqiang, Mpofu Enock, Tu Tingyong, Huai Qiangqiang, Sun Jiaowen, Chen Shanshan, Ding Jian, Shi Zhongping.Transcriptional analysis for carbon metabolism and kinetic modeling for heterologous proteins productions by Pichia pastoris in induction process with methanol/sorbitol co-feeding.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.05.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Transcriptional analysis for carbon metabolism and kinetic modeling for heterologous proteins productions by Pichia pastoris in induction process with methanol/sorbitol co-feeding Luqiang Jia1, Enock Mpofu2, Tingyong Tu1, Qiangqiang Huai1, Jiaowen Sun1, Shanshan Chen1, Jian Ding1*, Zhongping Shi1** 1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 214122 Wuxi, China 2. Department of Food Processing Technology, Harare Institute of Technology, Harare, Zimbabwe *
Corresponding School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122,
China. Tel/Fax: +86-510-85326276; Email: [email protected]. **
Corresponding authors: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi
214122, China. Tel/Fax: +86-510-85918292; Email: [email protected]
1
Highlights
1. “Methanol sufficient-oxygen limited” enhances the protein synthesis of MutS strain.
2. “Methanol sufficient-oxygen limited” enhances methanol metabolism in MutS strain.
3. “Methanol sufficient-oxygen limited” represses the protein synthesis of Mut+ strain.
4. “Methanol sufficient-oxygen limited” doesn’t affect methanol metabolism in Mut+ strain.
5. Models for the relationship between methanol and sorbitol utilization were designed.
Abstract: It is difficult to control concentrations of methanol/dissolved oxygen at high levels simultaneously in heterologous proteins productions by Pichia pastoris during induction phase. Two strains, a methanol utilization slow (MutS) type and a plus (Mut+) type were used with methanol/sorbitol co-feeding strategy to induce porcine interferon-α and human serum albumin-human granulocyte colony stimulating factor respectively, under the conditions of “methanol sufficientoxygen limited (MS-OL)” and “methanol limited-oxygen sufficient (ML-OS)”. For the MutS/Mut+ strains, the target proteins titers under “MS-OL” were 6-fold/19.2% of those under “ML-OS”. The key genes in methanol metabolism of the MutS strain were up-regulated under “MS-OL”, but they were not differently expressed in the Mut+ strain. Methanol utilization rate (rMeOH) of the MutS strain reduced when decreasing methanol concentration, but rMeOH of the Mut+ strain unchanged unless methanol concentration decreased to a low-limit of 0.6 g/L. Finally, kinetic models were designed to describe the methanol/sorbitol co-feeding process. Keywords: P. pastoris; methanol/sorbitol co-feeding; methanol concentration; dissolved oxygen; transcriptome analysis; kinetic model
2
1. Introduction The methylotrophic Pichia pastoris, the most effective and available systems for the expression of heterologous proteins, has the following advantages: it can be easily manipulated at the molecular genetic level; it can easily grow to high cells density and express proteins at high levels [1, 2]. Heterologous protein production by fed-batch culture with recombinant P. pastoris is basically divided into two phases: a growth phase to accumulate a large amount of functional cells, and an induction phase with methanol as inducer to produce heterologous protein [3]. Methanol/sorbitol co-feeding is an effective method in inducing heterologous protein by P. pastoris. Recently, it was reported that the addition of sorbitol could supply extra energy to cell and enhance both of cell growth and target protein productivity [4, 5]. In the induction with methanol/sorbitol co-feeding, no matter for MutS or Mut+ strain, simultaneously controlling concentrations of methanol and dissolved oxygen (DO) at high (proper) levels is very difficult, because metabolisms of methanol and oxygen couple with each other. However, controlling only one of them at high (proper) level is available. Methanol utilization slow (MutS) and methanol utilization plus (Mut+) are the two typical phenotypes of methylotrophic Pichia pastoris. In Mut+ strain, alcohol oxidase activity is extremely high, because two functional genes (aox1 and aox2) encode alcohol oxidase. In MutS strain, only aox2 genes exists, leading to the comparatively low alcohol oxidase activity. This is the most obvious difference between MutS and Mut+ strains. In previous research, it was found that MutS and Mut+ strains had different responses to the levels of methanol and oxygen supply: Mut+ strain effectively produced recombinant protein under “methanol limited and oxygen sufficient (ML-OS)” condition, but decreasingly produced recombinant protein under “methanol sufficient and oxygen limited (MS-OL)” condition [6]. Oppositely, high recombinant protein yield could be achieved under MS-OL condition for MutS strain [7]. However, except the 3
apparent induction performances, the physiological states in molecular level of MutS and Mut+ strains under the two standard conditions have seldom been concerned. Gene expression is a key event determining responses to environmental stimuli. Studying the whole genome at the transcriptional level may facilitate the elucidation of the molecular mechanisms of physiological processes in P. pastoris cell under ML-OS and MS-OL conditions. The transcriptome is the overall set of transcribed regions of the genome. Next-generation sequencing technologies are powerful strategies for identifying and quantifying gene expression at genome-wide level in unprecedented perspective [8, 9]. RNA sequencing (RNA-Seq) based on next-generation deep sequencing is the most powerful tool available for comparative transcriptome profiling [10, 11]. The analysis of transcriptional changes of P. pastoris may reveal specific genes involved in the regulation of physiological events under the aforementioned two induction conditions. In this work, the transcriptome analysis was used to compare cell physiological states under the MSOL and the ML-OS conditions. Based on the transcriptional analysis for carbon metabolism of P. pastoris, the MutS and Mut+ strains’ characteristics in utilizing methanol and sorbitol were speculated and verified. Finally, two kinetic models were designed to describe the methanol/sorbitol co-feeding processes of MutS and Mut+ P. pastoris. 2. Materials and methods 2.1 Microorganism A recombinant MutS strain (KM71), provided by Shanghai Academy of Agricultural Science, was used to express poircine interferon-α (pIFN-α). A recombinant Mut+ strain (GS115), provided by School of
4
Pharmaceutical Sciences, Jiangnan University, was used to express human serum albumin-human granulocyte colony stimulating factor (HSA-GCSFm). 2.2 Media The composition of the media for seed culture, fed-batch culture, feeding and induction was reported in our previous work [4]. 2.3 Heterologous protein expression by P. pastoris fed-batch cultivations The fed-batch culture was implemented in a 5 L bench-scaled fermenter (BLBIO-5GJ-3-H, Bailun Bio Co. China), with the initial batch media of 2.3 L. Inoculation and aeration rate were 13% (v/v) and 4 vvm. Temperature and pH were controlled at 30◦C and 6.0 during cell growth stage. The previously proposed improved DO-stat method [7] was used for feeding glycerol during growth stage, allowing cell density to reach 100~130 g-DWC/L. The induction phase was initiated by feeding methanol and sorbitol after glycerol was depleted. When fermentation was shifted into induction period, MS-OL and ML-OS conditions were achieved by the following “Strategy I” and “strategy II”, respectively. Strategy I: based on the on-line measurement of methanol electrode (FC-2002, Subo Co., China), the methanol concentration was maintained at 5 g/L with ON-OFF control manner, meanwhile sorbitol was co-fed with methanol/sorbitol feed ratio of 4:1 (g/g). Strategy II: DO was maintained at 10% (ODset = 10%) by regulating methanol feeding rate (F) with the equation: F=F*+Kc×(DO-DOset). The standard methanol feeding rate (F*) and the control parameter (Kc) were set at 0.7 mL·min-1 and 0.05, respectively. Sorbitol was also co-fed with methanol/sorbitol feed ratio of 4:1 (g/g). 2.4 Analytical methods
5
Cell density, concentrations of methanol and target proteins were off-line determined with the previously reported methods [12]. Formate concentration in cell was determined with the previously reported methods [4]. The transcriptome analysis based on RNA-seq was entrusted to Novogene Co., and its steps are the same with the report [13]. The differences of the genes expressed levels are described with the regulated ratio (R) defined by Eq (1), where L1 and L2 are the expressed levels of the specific gene under the MS-OL and ML-OS conditions respectively. The R value higher than 0.5850 indicates that the specific gene is up-regulated under MS-OL condition; the R value lower than -0.5850 indicates that the specific gene is down-regulated under MS-OL condition; the R value in the range of -0.5850~0.5850 indicates that the specific gene is not differently expressed.
R log 2
L1 L2
(1)
3 Results and discussion 3.1 Induction performances of the MutS and Mut+ strains under MS-OL and ML-OS conditions Methanol and oxygen are the most significant factors in the heterologous protein production by P. pastoris. In the induction processes with methanol/sorbitol co-feeding, simultaneously controlling concentrations of methanol and DO at high (proper) levels is very difficult, but controlling only one of them, meaning MS-OL or ML-OS condition, is available. In the induction with methanol/sorbitol co-feeding, target protein could be effectively expressed when methanol concentration was higher than 5 g/L [7]. Therefore, the condition with a methanol concentration higher than 5 g/L was considered as methanol sufficient (MS). DO is a crucial factor for foreign expression by P. pastoris. However, there are no clear classifications regarding to high (fully aerobic) and low (anaerobic) DO concentrations in foreign protein expression related literatures. In general, in most of heterologous protein production 6
by P. pastoris, DO is kept between 10% and 30% to maintain aerobic environment [14, 15]. Thus, in this study, we considered that 10% DO could be categorized to be the “aerobic classification”, meaning the oxygen sufficient (OS) condition. pIFN-α production by the MutS strain was implemented with MS-OL and ML-OS conditions respectively. Under MS-OL condition, methanol concentration was controlled at 5~10 g/L with DO at 0% in most induction phase; under ML-OS condition, OD was controlled at 10~15% with methanol concentration lower than 1 g/L in most induction phase (Fig. 1A). In the two induction processes, sorbitol was not excessively accumulated in broth, and its concentrations were still lower than 3 g/L (Fig. 1C). The final pIFN-α yield under MS-OL condition was 6-fold of that under ML-OS condition (Fig. 2A and 2C). Meanwhile, HSA-GCSFm production by the Mut+ strain was also implemented with the two conditions. The MS-OL and the ML-OS conditions were achieved (Fig. 1B), and almost no sorbitol was accumulated in broth (Fig. 1D). The final HSA-GCSFm yield under ML-OS condition was 5.2-fold of that under MS-OL condition (Fig. 2B and 2D). The results indicate that the ML-OS condition is superior for the protein production of Mut+ strain, and the MS-OL condition is superior for the protein production of MutS strain. It is widely known that the formate accumulated in P.pastoris cell may reduce cell viability and repress protein expression. The formate accumulated in cell and secreted into supernatant of each runs were actually measured. The former was at very low level of 0.05 g/g-DCW (Fig.1E and 1F) while the latter could not be detected. Fig.1 and Fig. 2 Besides the MS-OL and the ML-OS, “methanol-sufficient with oxygen-sufficient condition” could be considered as another optional condition for induction with methanol/sorbitol co-feeding. This 7
condition was not investigated due to the following reasons: 1) Oxygen consumption couples with methanol consumption, simultaneously controlling methanol concentration and DO at each individual “sub-optimal” level is very difficult. 2) “methanol-sufficient with oxygen-sufficient condition” could be realized when aerating pure oxygen. Because of the high sensitivity of DO in response to aeration and agitation changes when pure oxygen is used, severe fluctuation of DO can not be avoided [6], which deteriorates the expression of target protein [16, 17]. 3.2 Expressed levels of the key genes in carbon metabolisms of the MutS and Mut+ strains The transcriptome analysis for the MutS and Mut+ strains was achieved respectively, to compare the physiological states of cell under the MS-OL and the ML-OS conditions. For the MutS strain, 504 differently expressed genes (DEGs) were determined in the run with MS-OL condition versus the run with ML-OS condition, including 257 up-regulated genes and 247 down-regulated genes. For the Mut+ strain, 512 DEGs were determined in the run with MS-OL condition versus the run with ML-OS condition, including 235 up-regulated genes and 277 down-regulated genes. The DEGs in methanol and sorbitol metabolisms and the values of their regulated ratios were shown in Fig. 3. In addition, all of the DEGs in metabolic pathways were listed in Appendix 1. Summarizing the major results in Fig.3 and Appendix 1, the following conclusions could be achieved: 1) the increased pIFN-α production by MutS strain under the MS-OL condition was due to the enhanced methanol metabolism and biosynthesis of peroxisome/ribosomal proteins; 2) the decreased HSA-GCSFm production by Mut+ strain under the MS-OL condition was caused by the repressed H2O2 degrade and the enhanced formation of unfolded/mis-folded proteins.
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In the methanol/sorbitol co-feeding process, feeding rates of methanol and sorbitol are the two crucial operating variables affecting the expression of recombinant protein. Accordingly, the present study focused on the characteristics of methanol and sorbitol metabolisms under the two standard conditions, with the purpose of supplying guidance for the optimal control of methanol and sorbitol feeding. For the MutS strain producing pIFN-α, several key genes in methanol metabolism (AOX, FLD, DAS, TAL, RPIA and F16P) and respiratory chain (ATP synthase and complex IV) were up-regulated by MS-OL condition, but key genes in sorbitol metabolism were not differently expressed (Fig. 3). However, for the Mut+ strain producing HSA-GCSFm, several key genes in sorbitol metabolism (CISY, ACON, IDH, OGDC, SUCA, SDHA, FUM, MDHC) were down-regulated under MS-OL condition, and the key genes in methanol metabolism were not differently expressed (Fig. 3). The MutS strain had upregulated AOX and FLD genes and an unchanged FDH gene under the MS-OL condition. As a result, it seems that formate accumulation was more likely to happen under the MS-OL condition. However, actually, the intracellular accumulation of formate under the MS-OL was not higher than that under the ML-OS condition (Fig. 1E and 1F), which was due to the low carbon metabolism load in methanol dissimilation caused by methanol/sorbitol co-feeding [4, 6]. Fig. 3 3.3 MutS and Mut+ strains’ characteristics of utilizing methanol and sorbitol MS-OL condition enhanced the expression of the genes in methanol metabolism in the MutS strain. Therefore, it was speculated that methanol consumption rate of the MutS strain under the MS-OL condition was much more than that under the ML-OS condition. Oppositely, the genes in methanol metabolism of the Mut+ strain were not up-regulated by MS-OL condition. Therefore, it was speculated 9
that the methanol consumption rates of the Mut+ strain under the MS-OL and the ML-OS conditions should not be obviously different. The above speculations are proved by Fig. 4. For the MutS strain, average methanol consumption rate (rMeOH,ave) under the MS-OL condition was 50.49% higher than that under the ML-OS condition (Fig. 4A). On the other hand, rMeOH,ave of the Mut+ strain under the MS-OL condition is 9.81% higher than that under the ML-OS condition (Fig. 4B). Although the key genes in sorbitol metabolism in the Mut+ strain was down-regulated by the MS-OL condition, average sorbitol consumption rate (rSor,ave) did not reduced compared to that under the ML-OS condition. As described in the 2.3 section, the ratio between methanol and sorbitol feeding rate was 4:1. With the low feeding rate, sorbitol was not excessively accumulated in broth for all the runs (Fig. 1C and 1D). Therefore, consumption rate of sorbitol was limited by its feeding rate, but not the expression levels of the key genes in sorbitol metabolism. Fig. 4 and Fig. 5 To further investigate the two strains’ characteristics of utilizing methanol, two experiments were designed and implemented for the MutS and Mut+ strains respectively. In the experiments, methanol depletion were purposely made, the variations of methanol and DO concentration in the processes were monitored on-line and shown in Fig. 5A and 5B. As methanol metabolism and oxygen metabolism couple with each other, an increased DO indicates a decreased methanol consumption rate when other conditions are maintained at constant levels. For the presently used MutS strain, the smoothly increased DO indicated a slowly and continuously decreased methanol consumption rate (Fig. 5A). For the Mut+ strain, the suddenly increased DO indicated a steeply decreased methanol consumption rate (Fig. 5B). Accordingly, the curves of methanol consumption rate versus methanol concentration were illustrated in Fig. 5C and 5D. The results revealed that the methanol consumption rate (rMeOH) of the MutS strain 10
relied on the methanol concentration, low methanol concentration repressed the utilization of methanol. Meanwhile, rMeOH of the Mut+ strain did not rely on the methanol concentration, meaning that rMeOH was a constant only if the methanol concentration above a limit level. The limit level for the used Mut+ strain was about 0.6 g/L (red dash line in Fig. 5B and 5D). In Fig. 4, it was observed that the rMeOH,ave of the MutS strain was much lower than that of the Mut+ strain under the same operation condition. This is because the natures or differences of AOX promoters in MutS and the Mut+ strains. Mut+ strain contains both aox1 and aox2 genes. The functional copy of the aox1 gene accounts for approximately 85% AOX activity, while aox2 gene accounts for the rest 15% of AOX activity [18]. On the other hand, MutS strain does not have the aox1 gene and its AOX activity completely relies on the aox2 gene, thus, MutS strain has a much lower AOX activity and methanol consumption ability than those of Mut+ strain [18, 19]. In this study, sorbitol was co-fed with methanol at a fixed feeding ratio (1:4, g/g), as a result, methanol and sorbitol consumption rates were higher for Mut+ as compared to MutS. 3.4 Kinetic models for methanol and sorbitol utilization Methanol/sorbitol co-feeding for P. pastoris is the process with the following features: 1) carbon is supplied by methanol and sorbitol together, and total carbon consumption rate is a constant at a specific time; 2) methanol (primary carbon source) feeding rate should be relatively high for enough inducing intensity; 3) sorbitol (assisted carbon source) feeding rate should be relatively low and sorbitol should not accumulated in broth, or the methanol consumption rate will decrease, which weakens the inducing intensity [6]. Based on the features, Eq. (2) was designed to describe the relationship between methanol and sorbitol utilization. SMeOH and SF,MeOH are the methanol concentrations in broth and the methanol 11
feeding media; SF,Sor is the sorbitol concentration in the sorbitol feeding media; FMeOH is methanol feeding rate; FSor is sorbitol feeding rate, which is equivalent to the sorbitol consumption rate. R*Sor and n are the parameters of the model. RMeOH is the absolute methanol consumption rate without regarding to the effect of sorbitol addition. According to the characteristics shown in Fig. 5C and 5D, RMeOH for MutS and Mut+ strains could be calculated by Eq. (3) and Eq. (4) respectively, where R*MeOH, K, C and Slimit are parameters of the model. In the future work, a number of runs with different methanol and sorbitol feeding rates will be carried out to obtain modeling data. After evaluating all of the parameters, the kinetic models could be used to describe the methanol/sorbitol co-feeding process of P. pastoris for producing different heterologous proteins. n F S dS MeOH Sor F,Sor FMeOH S F,MeOH RMeOH 1 * (2) dt RSor
RMeOH
* RMeOH S MeOH K S MeOH
(3)
C S MeOH Slimit RMeOH 0 S MeOH Slimit
(4)
4. Conclusion Transcriptional levels of genes in carbon metabolism of a MutS strain and a Mut+ strain under the two extreme conditions of “methanol sufficient-oxygen limited (MS-OL)” and “methanol limited-oxygen sufficient (ML-OS)” were compared. The results revealed that, methanol metabolism of the MutS strain was enhanced under “MS-OL”, while the intensity of methanol metabolism of the Mut+ strain under the two conditions were similar. Subsequently, methanol and sorbitol utilizations in the MutS and Mut+ strains were characterized as: methanol utilization rate of the MutS strain gradually decreased when 12
decreasing methanol concentration; methanol utilization rate of the Mut+ strain unchanged unless methanol concentration decreased to a low-limit. Finally, the above characteristics were described with the developed kinetic models. Acknowledgments The authors thank the financial supports from Natural Science Foundation of China (#21606106), Natural Science Foundation of Jiangsu Province (#BK20150127, #BK20160162), the 111 Project (#111-2-06) of China, the Fundamental Research Funds for the Central Universities (JUSRP51632A, JUSRP11536) and Industry-Education-Research Cooperation Project of Jiangsu Province (BY2016022-15). Reference [1]
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Appendix 1 DEGs selected between MS-OL and ML-OS concentration in MutS P. pastoris DEGs selected in methanol metabolism Gene
Gene function
Regulate
Log2Ratio
AOX2
Alcohol oxidase2
UP
0.97±0.17
FLD
Formaldehyde dehydrogenase
UP
1.73±0.71
DAS
Dihydroxyacetone synthase
UP
1.29±0.29
F16P
Fructose-1,6-bisphosphatase
UP
0.89±0.35
RPIA
Ribose-5-phosphate isomerase
UP
0.78±0.05
TAL1
Transaldolase
UP
0.88±0.12
DEGs selected peroxisome biosynthesis metabolism Gene
Gene function
Regulate
Log2Ratio
PEX13
Peroxin13
UP
0.88±0.26
PMP34
Peroxisomal membrane protein34
UP
0.75±0.04
PEX2
Peroxin2
UP
0.72±0.17
PEX3
Peroxin3
UP
0.74±0.08
AGX1
Alanine-glyoxylate aminotransferase
UP
0.80±0.10
PEX5
Peroxin5
UP
0.75±0.16
CAT
Catalase
UP
0.82±0.07
DEGs selected related to respiratory chain Gene
Gene function
Regulate
Log2Ratio
ComplexⅣ5b
Cytochrome c oxidase subunit 5b
UP
0.79±0.18
ComplexⅣ6b
Cytochrome c oxidase subunit 6b
UP
0.85±0.22
ATP synthase
Mitochondrial ATP synthase
UP
0.98±0.27
DEGs related to ribosomal proteins biosynthesis metabolism Gene
Gene function
Regulate
RS6
40S ribosomal protein S6
UP
0.94±0.35
RS27B
40S ribosomal protein S27-B
UP
0.73±0.21
RS21_
40S ribosomal protein S21
UP
0.82±0.17
16
Log2Ratio
RLA0
60S ribosomal protein
UP
0.71±0.08
RL15
60S ribosomal protein L15
UP
1.02±0.32
RS26B
40S ribosomal protein S26-B
UP
0.98±0.11
RL22A
60S ribosomal protein L22-A
UP
1.15±0.17
RS12
40S ribosomal protein S12
UP
0.87±0.22
RS10A
40S ribosomal protein S10-A
UP
1.12±0.09
RS13
40S ribosomal protein S13
UP
0.93±0.20
RS22
40S ribosomal protein S22
UP
0.91±0.06
RS4B
40S ribosomal protein S4-B
UP
0.66±0.13
RL30
60S ribosomal protein L30
UP
0.89±0.31
RL17
60SribosomalproteinL17
UP
0.74±0.09
RS7B_
40S ribosomal protein S7-B
UP
0.96±0.19
RL1B
60S ribosomal protein L1-B
UP
0.92±0.40
RS3A
40S ribosomal proteinS1
UP
0.91±0.14
RS11B
40S ribosomal proteinS11-B
UP
0.89±0.23
RL6B
60S ribosomal protein L6-B
UP
0.73±0.12
RL44
60S ribosomal protein L44
UP
1.02±0.39
DEGs selected between MS-OL and ML-OS concentration in Mut+ P. pastoris DEGs selected in TCA circle Gene
Gene fuction
Regulate
Log2Ratio
PCKA
Phosphoenolpyruvate carboxykinase
DOWN
-1.71±0.11
ACS1
Acetyl-coenzyme A synthetase 1
DOWN
-2.41±0.28
OGDC
2-oxoglutarate dehydrogenase E1
DOWN
-0.78±0.13
IDH2
componentdehydrogenase 2Isocitrate
DOWN
-1.8±0.32
SUCA
oxoglutarate E1 Succinyl-CoAdehydrogenase synthetase
DOWN
-0.63±0.09
CISY
Citrate synthase,
DOWN
-2.56±0.47
FUM
Fumarase
DOWN
-0.87±0.04
17
SDHA
Succinate dehydrogenase
DOWN
-0.94±0.18
ACON
Aconitase
DOWN
-2.17±0.75
DHDL
Dihydrolipoamide dehydrogenase
DOWN
-0.72±0.15
MDHC
Malate dehydrogenase
DOWN
-1.66±0.34
DOWN
-0.76±0.22
DOWN
-1.03±0.37
DOWN
-1.32±0.41
DEGs related to respiratory chain ComplexⅡ
Succinate dehydrogenase cytochrome b560 subunit
ComplexⅡ
Succinate dehydrogenase cytochrome b small subunit
ComplexⅡ
Succinate dehydrogenase flavoprotein subunit
DEGs select in amino acids biosynthesis Genes related to Val, Leu and Ile biosynthesis EC
Threonine dehydratase
UP
0.62±0.13
ILVB
Acetolactate synthase
UP
0.77±0.21
HADH
Dihydroxyacid dehydratase
UP
0.62±0.06
BCA1
Branched-chain-amino-acid
UP
0.65±0.16
UP
0.67±0.07
aminotransferase Genes related to Phe, Tyr and Try biosynthesis AROG
3-deoxy-D-arabino-heptulosonate-7phosphate (DAHP) synthase
CHMU
Chorismate mutase
UP
0.82±0.18
AATC
aspartate aminotransferase
UP
1.32±0.39
HIS8
Histidinol-phosphate aminotransferase
UP
0.88±0.12
TRP
Tryptophan synthase
UP
0.64±0.08
Genes related to Ser, Gly and Cys biosynthesis SERA
D-3-phosphoglycerate dehydrogenase
UP
0.78±0.06
CBS
Cystathionine beta-synthase
UP
0.82±0.11
DEGs selected in fatty acid degradation metabolism Gene
Gene fuction
regulate
Log2Ratio
LCF1
Long chain fatty acyl-CoA synthetase1
DOWN
-1.59±0.43
18
PXA2
Peroxisomal long-chain fatty acid import protein 1
DOWN
-2.95±0.66
LCF2
Long chain fatty acyl-CoA synthetase 2
DOWN
-3.27±0.47
PXA1
Peroxisomal long-chain fatty acid import protein 2
DOWN
-3.16±0.77
THIK
3-ketoacyl-CoA thiolase
DOWN
-2.43±0.73
CACP
O-acetyltransferase
DOWN
-2.78±0.49
PXMP4
Peroxisomal membrane protein 4
DOWN
-1.55±0.67
SODM
Superoxide dismutase [Mn]
DOWN
-0.78±0.28
ACOX
Acyl-coenzyme A oxidase
DOWN
-3.22±0.84
PMP20
peroxiredoxin pmp20
DOWN
-0.90±0.13
SPS19
Peroxisomal 2,4-dienoyl-CoA reductase
DOWN
-1.33±0.34
DEGs in ubiquitin-proteasome system Gene
Gene function
Regulate
RPN13
26S proteasome regulatory subunit RPN13
UP
0.63±0.21
PSB3
Proteasome subunit beta type-3
UP
0.80±0.10
RPN11
26S proteasome regulatory subunit RPN11
UP
0.70±0.29
PRS6B
26S protease regulatory subunit 6B
UP
0.65±0.12
RPN2
26S proteasome regulatory subunit RPN2
UP
0.77±0.21
PSB5
Proteasome subunit beta type-5
UP
0.83±0.11
CDC48
ATPase in ER
UP
0.73±0.17
PDI
Protein disulfide-isomerase
UP
1.17±0.25
OSTB
Dolichyl-diphosphooligosaccharide--protein
UP
0.64±0.08
PNG1
Peptide-N(4)- asparagine amidase
UP
0.59±0.14
Unknown
Ubiquitin ligase complex domain-containing proteins
UP
0.60±0.09
SC61B
Protein transport protein SEC61 subunit beta
UP
0.67±0.10
SC61G
Protein transport protein SEC61 subunit gamma
UP
1.08±0.37
19
Log2ratio
Figure captions Fig. 1 Variations of concentrations of DO, methanol, sorbitol, cell and formate in cell for the MutS and Mut+ strains under the MS-OL and the ML-OS conditions. A, C and E: the results for the production of pIFN-α by the MutS strain; B, D and F: the results for the production of HSA-GCSFm by the Mut+ strain. Filled squares: methanol concentrations under MS-OL condition; open squares: methanol concentrations under ML-OS condition; filled triangles: sorbitol concentrations under MS-OL condition; open triangles: sorbitol concentrations under ML-OS condition; filled circles: cell concentrations under MS-OL condition; open circles: cell concentrations under ML-OS condition; filled diamonds: formate concentrations in cell under MS-OL condition; open diamonds: formate concentrations in cell under ML-OS condition; solid line: DO under MS-OL condition; dash line: DO under ML-OS condition. Fig. 2 Induction performances of the MutS and Mut+ strains under MS-OL and ML-OS conditions. A: variation of pIFN-α concentration in induction phase; B: variation of HSA-GCSFm concentration in induction phase; C: result of SDS-PAGE for the pIFN-α production process; MutS strain; D: result of SDS-PAGE for the HSA-GCSFm production process. Filled circles: target protein concentrations under the MS-OL condition; open circles: target protein concentrations under the ML-OS condition. Fig. 3 Comparison of the expression levels of the key genes in carbon (methanol and sorbitol) metabolism. a: MutS; b: Mut+. Fig. 4 Average methanol and sorbitol consumption rates of the MutS and Mut+ strains under MS-OL and ML-OS conditions. A: result for the MutS strain; B: result for the Mut+ strain.
20
Fig. 5 The MutS and Mut+ strains’ characteristics of utilizing methanol. A and B showed the variations of methanol concentrations in the period when methanol was gradually running out. C and D showed the variations of methanol consumption rates versus methanol concentrations. A and C: result for the MutS strain; B and D: result for the Mut+ strain. Solid lines: methanol concentrations; black dash lines: DO; red dash lines: limit levels of methanol concentration; open circles: methanol consumption rates.
21
6 40 4 2
0
0 0
80
10 8
60
6 40 4 20
2 0
0
10 20 30 40 50 60 70
0
10 20 30 40 50 60 70
Fermentation time after induction (h)
Fermentation time after induction (h)
20
20
Sorbitol conc. (g/L)
C
15 10 5
D
15 10 5 0
0 10 20 30 40 50 60 70
0
Fermentation time after induction (h) 0.2
120 100 80
0.1
60 40 20 0.0
0 0
10 20 30 40 50 60 70
140
Cell conc. (g/L)
E
Formate conc. (g/g-DCW)
140
10 20 30 40 50 60 70
Fermentation time after induction (h) F
120 100 80
0.1
60 40 20 0.0
0 0
Fermentation time after induction (h)
0.2
10 20 30 40 50 60 70
Fermentation time after induction (h)
Fig.1
22
Formate conc. (g/g-DCW)
0
Cell conc. (g/L)
B
Methanol conc. (g/L)
8
60
20
Sorbitol conc. (g/L)
10
DO (%)
A
Methanol conc. (g/L)
DO (%)
80
0.8
A
HAS-GCSF conc. (g/L)
1.5 1.0
m
pIFN- conc. (g/L)
2.0
0.5 0.0
B
0.6 0.4 0.2 0.0
0 10 20 30 40 50 60 70 Fermentation time after induction (h) C
MS-OL
0 10 20 30 40 50 60 70 Fermentation time after induction (h)
ML-OS
ML-OS
D
M 8h 32h 56h 70h 8h 32h 56h 70h
MS-OL
M 8h 32h 56h 70h 8h 32h 56h 70h 150KD
66KD 45KD 35KD 27KD 20KD 14.4KD 9.5KD 6.5KD
100KD 70KD 50KD
HSA-GCSFm 85KD
35KD
pIFN-α 25KD 16KD 20KD 15KD
Fig.2
23
Sor SDH F6P a:ND/b:ND
O2
CH3OH
GAP
Pathway B CH3OH
PYR
Pathway C
O2
Fatty acid AcCoATHIK a:ND/b:-5.38
H2O2
AOX a:0.97/b:ND HCHO
CISY OAA CIT a:ND/b:-2.55 MDHC ACON a:ND/b:-1.65 a:ND/b:-2.17 MAL ICIT FUM a:ND/b:-0.87
TCA
FUM SDHA a:ND/b:-0.94 SUC
HCHO
TAL DHA GAP a:0.97/b:ND R5P Peroxisome
IDH a:ND/b:-1.79 α-KG OGDC a:ND/b:-0.78 Suc-COA
DHA
ATP ADP DHAP GAP
R5P F16P a:0.89/b:ND FBP F6P
Pathway A Targeted Protein
Functional cell
Pi
NADH
FAD
ComplexⅡ a: ND/b: -1.32
CO2
Xy5P
NAD+ ADP ATP synthase a: 0.98/b: ND ATP CoQ ComplexⅢ
NADH 2eComplexⅠ FADH2 2e-
CO2
DAS a:1.29/b:ND
SUCA a:ND/b:-0.63
FADH2
FLD a: 1.73/b:ND FDH HCOOH GS-CH2OH + NAD NAD+ NADH NADH GSH
ComplexⅣ - a: 0.85/ND Cytochrome c 2e 1/2O2+2H+ ATP synthase ATP synthase ADPa: 0.98/b: ND ATP ADP a: 0.98/b: NDATP
Fig. 3
24
H2O
A
10
Methanol Sorbitol
8 6 4 2 0
12
rMeOH, ave, rSor, ave (g/L/h)
rMeOH, ave, rSor, ave (g/L/h)
12
B
10
Methanol Sorbitol
8 6 4 2 0
MS-OL
ML-OS
MS-OL
Fig. 4
25
ML-OS
80
0.6
60
0.3
40
2.0
0.5
1.0 1.5 Time (h)
2.0
4
20 2.5
80
2
60
1
40
5
0.5
1.0 1.5 Time (h)
2.0
20 2.5
D
4
rMeOH (g/L/h)
1.5 1.0 0.5 0.0 0.0
3
0 0.0
C
100
methanol addition
B
3 2 1 0
0.3 0.6 0.9 1.2 Methanol concentration (g/L)
0
Fig. 5
26
1 2 3 4 Methanol concentration (g/L)
DO (%)
0.9
0.0 0.0
rMeOH (g/L/h)
100
A
DO (%) Methanol conc. (g/L)
Methanol conc. (g/L)
1.2
S1359-5113(17)30153-8 http://dx.doi.org/doi:10.1016/j.procbio.2017.05.011 PRBI 11040
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
23-1-2017 6-5-2017 10-5-2017
Please cite this article as: Jia Luqiang, Mpofu Enock, Tu Tingyong, Huai Qiangqiang, Sun Jiaowen, Chen Shanshan, Ding Jian, Shi Zhongping.Transcriptional analysis for carbon metabolism and kinetic modeling for heterologous proteins productions by Pichia pastoris in induction process with methanol/sorbitol co-feeding.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.05.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Transcriptional analysis for carbon metabolism and kinetic modeling for heterologous proteins productions by Pichia pastoris in induction process with methanol/sorbitol co-feeding Luqiang Jia1, Enock Mpofu2, Tingyong Tu1, Qiangqiang Huai1, Jiaowen Sun1, Shanshan Chen1, Jian Ding1*, Zhongping Shi1** 1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 214122 Wuxi, China 2. Department of Food Processing Technology, Harare Institute of Technology, Harare, Zimbabwe *
Corresponding School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122,
China. Tel/Fax: +86-510-85326276; Email: [email protected]. **
Corresponding authors: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi
214122, China. Tel/Fax: +86-510-85918292; Email: [email protected]
1
Highlights
1. “Methanol sufficient-oxygen limited” enhances the protein synthesis of MutS strain.
2. “Methanol sufficient-oxygen limited” enhances methanol metabolism in MutS strain.
3. “Methanol sufficient-oxygen limited” represses the protein synthesis of Mut+ strain.
4. “Methanol sufficient-oxygen limited” doesn’t affect methanol metabolism in Mut+ strain.
5. Models for the relationship between methanol and sorbitol utilization were designed.
Abstract: It is difficult to control concentrations of methanol/dissolved oxygen at high levels simultaneously in heterologous proteins productions by Pichia pastoris during induction phase. Two strains, a methanol utilization slow (MutS) type and a plus (Mut+) type were used with methanol/sorbitol co-feeding strategy to induce porcine interferon-α and human serum albumin-human granulocyte colony stimulating factor respectively, under the conditions of “methanol sufficientoxygen limited (MS-OL)” and “methanol limited-oxygen sufficient (ML-OS)”. For the MutS/Mut+ strains, the target proteins titers under “MS-OL” were 6-fold/19.2% of those under “ML-OS”. The key genes in methanol metabolism of the MutS strain were up-regulated under “MS-OL”, but they were not differently expressed in the Mut+ strain. Methanol utilization rate (rMeOH) of the MutS strain reduced when decreasing methanol concentration, but rMeOH of the Mut+ strain unchanged unless methanol concentration decreased to a low-limit of 0.6 g/L. Finally, kinetic models were designed to describe the methanol/sorbitol co-feeding process. Keywords: P. pastoris; methanol/sorbitol co-feeding; methanol concentration; dissolved oxygen; transcriptome analysis; kinetic model
2
1. Introduction The methylotrophic Pichia pastoris, the most effective and available systems for the expression of heterologous proteins, has the following advantages: it can be easily manipulated at the molecular genetic level; it can easily grow to high cells density and express proteins at high levels [1, 2]. Heterologous protein production by fed-batch culture with recombinant P. pastoris is basically divided into two phases: a growth phase to accumulate a large amount of functional cells, and an induction phase with methanol as inducer to produce heterologous protein [3]. Methanol/sorbitol co-feeding is an effective method in inducing heterologous protein by P. pastoris. Recently, it was reported that the addition of sorbitol could supply extra energy to cell and enhance both of cell growth and target protein productivity [4, 5]. In the induction with methanol/sorbitol co-feeding, no matter for MutS or Mut+ strain, simultaneously controlling concentrations of methanol and dissolved oxygen (DO) at high (proper) levels is very difficult, because metabolisms of methanol and oxygen couple with each other. However, controlling only one of them at high (proper) level is available. Methanol utilization slow (MutS) and methanol utilization plus (Mut+) are the two typical phenotypes of methylotrophic Pichia pastoris. In Mut+ strain, alcohol oxidase activity is extremely high, because two functional genes (aox1 and aox2) encode alcohol oxidase. In MutS strain, only aox2 genes exists, leading to the comparatively low alcohol oxidase activity. This is the most obvious difference between MutS and Mut+ strains. In previous research, it was found that MutS and Mut+ strains had different responses to the levels of methanol and oxygen supply: Mut+ strain effectively produced recombinant protein under “methanol limited and oxygen sufficient (ML-OS)” condition, but decreasingly produced recombinant protein under “methanol sufficient and oxygen limited (MS-OL)” condition [6]. Oppositely, high recombinant protein yield could be achieved under MS-OL condition for MutS strain [7]. However, except the 3
apparent induction performances, the physiological states in molecular level of MutS and Mut+ strains under the two standard conditions have seldom been concerned. Gene expression is a key event determining responses to environmental stimuli. Studying the whole genome at the transcriptional level may facilitate the elucidation of the molecular mechanisms of physiological processes in P. pastoris cell under ML-OS and MS-OL conditions. The transcriptome is the overall set of transcribed regions of the genome. Next-generation sequencing technologies are powerful strategies for identifying and quantifying gene expression at genome-wide level in unprecedented perspective [8, 9]. RNA sequencing (RNA-Seq) based on next-generation deep sequencing is the most powerful tool available for comparative transcriptome profiling [10, 11]. The analysis of transcriptional changes of P. pastoris may reveal specific genes involved in the regulation of physiological events under the aforementioned two induction conditions. In this work, the transcriptome analysis was used to compare cell physiological states under the MSOL and the ML-OS conditions. Based on the transcriptional analysis for carbon metabolism of P. pastoris, the MutS and Mut+ strains’ characteristics in utilizing methanol and sorbitol were speculated and verified. Finally, two kinetic models were designed to describe the methanol/sorbitol co-feeding processes of MutS and Mut+ P. pastoris. 2. Materials and methods 2.1 Microorganism A recombinant MutS strain (KM71), provided by Shanghai Academy of Agricultural Science, was used to express poircine interferon-α (pIFN-α). A recombinant Mut+ strain (GS115), provided by School of
4
Pharmaceutical Sciences, Jiangnan University, was used to express human serum albumin-human granulocyte colony stimulating factor (HSA-GCSFm). 2.2 Media The composition of the media for seed culture, fed-batch culture, feeding and induction was reported in our previous work [4]. 2.3 Heterologous protein expression by P. pastoris fed-batch cultivations The fed-batch culture was implemented in a 5 L bench-scaled fermenter (BLBIO-5GJ-3-H, Bailun Bio Co. China), with the initial batch media of 2.3 L. Inoculation and aeration rate were 13% (v/v) and 4 vvm. Temperature and pH were controlled at 30◦C and 6.0 during cell growth stage. The previously proposed improved DO-stat method [7] was used for feeding glycerol during growth stage, allowing cell density to reach 100~130 g-DWC/L. The induction phase was initiated by feeding methanol and sorbitol after glycerol was depleted. When fermentation was shifted into induction period, MS-OL and ML-OS conditions were achieved by the following “Strategy I” and “strategy II”, respectively. Strategy I: based on the on-line measurement of methanol electrode (FC-2002, Subo Co., China), the methanol concentration was maintained at 5 g/L with ON-OFF control manner, meanwhile sorbitol was co-fed with methanol/sorbitol feed ratio of 4:1 (g/g). Strategy II: DO was maintained at 10% (ODset = 10%) by regulating methanol feeding rate (F) with the equation: F=F*+Kc×(DO-DOset). The standard methanol feeding rate (F*) and the control parameter (Kc) were set at 0.7 mL·min-1 and 0.05, respectively. Sorbitol was also co-fed with methanol/sorbitol feed ratio of 4:1 (g/g). 2.4 Analytical methods
5
Cell density, concentrations of methanol and target proteins were off-line determined with the previously reported methods [12]. Formate concentration in cell was determined with the previously reported methods [4]. The transcriptome analysis based on RNA-seq was entrusted to Novogene Co., and its steps are the same with the report [13]. The differences of the genes expressed levels are described with the regulated ratio (R) defined by Eq (1), where L1 and L2 are the expressed levels of the specific gene under the MS-OL and ML-OS conditions respectively. The R value higher than 0.5850 indicates that the specific gene is up-regulated under MS-OL condition; the R value lower than -0.5850 indicates that the specific gene is down-regulated under MS-OL condition; the R value in the range of -0.5850~0.5850 indicates that the specific gene is not differently expressed.
R log 2
L1 L2
(1)
3 Results and discussion 3.1 Induction performances of the MutS and Mut+ strains under MS-OL and ML-OS conditions Methanol and oxygen are the most significant factors in the heterologous protein production by P. pastoris. In the induction processes with methanol/sorbitol co-feeding, simultaneously controlling concentrations of methanol and DO at high (proper) levels is very difficult, but controlling only one of them, meaning MS-OL or ML-OS condition, is available. In the induction with methanol/sorbitol co-feeding, target protein could be effectively expressed when methanol concentration was higher than 5 g/L [7]. Therefore, the condition with a methanol concentration higher than 5 g/L was considered as methanol sufficient (MS). DO is a crucial factor for foreign expression by P. pastoris. However, there are no clear classifications regarding to high (fully aerobic) and low (anaerobic) DO concentrations in foreign protein expression related literatures. In general, in most of heterologous protein production 6
by P. pastoris, DO is kept between 10% and 30% to maintain aerobic environment [14, 15]. Thus, in this study, we considered that 10% DO could be categorized to be the “aerobic classification”, meaning the oxygen sufficient (OS) condition. pIFN-α production by the MutS strain was implemented with MS-OL and ML-OS conditions respectively. Under MS-OL condition, methanol concentration was controlled at 5~10 g/L with DO at 0% in most induction phase; under ML-OS condition, OD was controlled at 10~15% with methanol concentration lower than 1 g/L in most induction phase (Fig. 1A). In the two induction processes, sorbitol was not excessively accumulated in broth, and its concentrations were still lower than 3 g/L (Fig. 1C). The final pIFN-α yield under MS-OL condition was 6-fold of that under ML-OS condition (Fig. 2A and 2C). Meanwhile, HSA-GCSFm production by the Mut+ strain was also implemented with the two conditions. The MS-OL and the ML-OS conditions were achieved (Fig. 1B), and almost no sorbitol was accumulated in broth (Fig. 1D). The final HSA-GCSFm yield under ML-OS condition was 5.2-fold of that under MS-OL condition (Fig. 2B and 2D). The results indicate that the ML-OS condition is superior for the protein production of Mut+ strain, and the MS-OL condition is superior for the protein production of MutS strain. It is widely known that the formate accumulated in P.pastoris cell may reduce cell viability and repress protein expression. The formate accumulated in cell and secreted into supernatant of each runs were actually measured. The former was at very low level of 0.05 g/g-DCW (Fig.1E and 1F) while the latter could not be detected. Fig.1 and Fig. 2 Besides the MS-OL and the ML-OS, “methanol-sufficient with oxygen-sufficient condition” could be considered as another optional condition for induction with methanol/sorbitol co-feeding. This 7
condition was not investigated due to the following reasons: 1) Oxygen consumption couples with methanol consumption, simultaneously controlling methanol concentration and DO at each individual “sub-optimal” level is very difficult. 2) “methanol-sufficient with oxygen-sufficient condition” could be realized when aerating pure oxygen. Because of the high sensitivity of DO in response to aeration and agitation changes when pure oxygen is used, severe fluctuation of DO can not be avoided [6], which deteriorates the expression of target protein [16, 17]. 3.2 Expressed levels of the key genes in carbon metabolisms of the MutS and Mut+ strains The transcriptome analysis for the MutS and Mut+ strains was achieved respectively, to compare the physiological states of cell under the MS-OL and the ML-OS conditions. For the MutS strain, 504 differently expressed genes (DEGs) were determined in the run with MS-OL condition versus the run with ML-OS condition, including 257 up-regulated genes and 247 down-regulated genes. For the Mut+ strain, 512 DEGs were determined in the run with MS-OL condition versus the run with ML-OS condition, including 235 up-regulated genes and 277 down-regulated genes. The DEGs in methanol and sorbitol metabolisms and the values of their regulated ratios were shown in Fig. 3. In addition, all of the DEGs in metabolic pathways were listed in Appendix 1. Summarizing the major results in Fig.3 and Appendix 1, the following conclusions could be achieved: 1) the increased pIFN-α production by MutS strain under the MS-OL condition was due to the enhanced methanol metabolism and biosynthesis of peroxisome/ribosomal proteins; 2) the decreased HSA-GCSFm production by Mut+ strain under the MS-OL condition was caused by the repressed H2O2 degrade and the enhanced formation of unfolded/mis-folded proteins.
8
In the methanol/sorbitol co-feeding process, feeding rates of methanol and sorbitol are the two crucial operating variables affecting the expression of recombinant protein. Accordingly, the present study focused on the characteristics of methanol and sorbitol metabolisms under the two standard conditions, with the purpose of supplying guidance for the optimal control of methanol and sorbitol feeding. For the MutS strain producing pIFN-α, several key genes in methanol metabolism (AOX, FLD, DAS, TAL, RPIA and F16P) and respiratory chain (ATP synthase and complex IV) were up-regulated by MS-OL condition, but key genes in sorbitol metabolism were not differently expressed (Fig. 3). However, for the Mut+ strain producing HSA-GCSFm, several key genes in sorbitol metabolism (CISY, ACON, IDH, OGDC, SUCA, SDHA, FUM, MDHC) were down-regulated under MS-OL condition, and the key genes in methanol metabolism were not differently expressed (Fig. 3). The MutS strain had upregulated AOX and FLD genes and an unchanged FDH gene under the MS-OL condition. As a result, it seems that formate accumulation was more likely to happen under the MS-OL condition. However, actually, the intracellular accumulation of formate under the MS-OL was not higher than that under the ML-OS condition (Fig. 1E and 1F), which was due to the low carbon metabolism load in methanol dissimilation caused by methanol/sorbitol co-feeding [4, 6]. Fig. 3 3.3 MutS and Mut+ strains’ characteristics of utilizing methanol and sorbitol MS-OL condition enhanced the expression of the genes in methanol metabolism in the MutS strain. Therefore, it was speculated that methanol consumption rate of the MutS strain under the MS-OL condition was much more than that under the ML-OS condition. Oppositely, the genes in methanol metabolism of the Mut+ strain were not up-regulated by MS-OL condition. Therefore, it was speculated 9
that the methanol consumption rates of the Mut+ strain under the MS-OL and the ML-OS conditions should not be obviously different. The above speculations are proved by Fig. 4. For the MutS strain, average methanol consumption rate (rMeOH,ave) under the MS-OL condition was 50.49% higher than that under the ML-OS condition (Fig. 4A). On the other hand, rMeOH,ave of the Mut+ strain under the MS-OL condition is 9.81% higher than that under the ML-OS condition (Fig. 4B). Although the key genes in sorbitol metabolism in the Mut+ strain was down-regulated by the MS-OL condition, average sorbitol consumption rate (rSor,ave) did not reduced compared to that under the ML-OS condition. As described in the 2.3 section, the ratio between methanol and sorbitol feeding rate was 4:1. With the low feeding rate, sorbitol was not excessively accumulated in broth for all the runs (Fig. 1C and 1D). Therefore, consumption rate of sorbitol was limited by its feeding rate, but not the expression levels of the key genes in sorbitol metabolism. Fig. 4 and Fig. 5 To further investigate the two strains’ characteristics of utilizing methanol, two experiments were designed and implemented for the MutS and Mut+ strains respectively. In the experiments, methanol depletion were purposely made, the variations of methanol and DO concentration in the processes were monitored on-line and shown in Fig. 5A and 5B. As methanol metabolism and oxygen metabolism couple with each other, an increased DO indicates a decreased methanol consumption rate when other conditions are maintained at constant levels. For the presently used MutS strain, the smoothly increased DO indicated a slowly and continuously decreased methanol consumption rate (Fig. 5A). For the Mut+ strain, the suddenly increased DO indicated a steeply decreased methanol consumption rate (Fig. 5B). Accordingly, the curves of methanol consumption rate versus methanol concentration were illustrated in Fig. 5C and 5D. The results revealed that the methanol consumption rate (rMeOH) of the MutS strain 10
relied on the methanol concentration, low methanol concentration repressed the utilization of methanol. Meanwhile, rMeOH of the Mut+ strain did not rely on the methanol concentration, meaning that rMeOH was a constant only if the methanol concentration above a limit level. The limit level for the used Mut+ strain was about 0.6 g/L (red dash line in Fig. 5B and 5D). In Fig. 4, it was observed that the rMeOH,ave of the MutS strain was much lower than that of the Mut+ strain under the same operation condition. This is because the natures or differences of AOX promoters in MutS and the Mut+ strains. Mut+ strain contains both aox1 and aox2 genes. The functional copy of the aox1 gene accounts for approximately 85% AOX activity, while aox2 gene accounts for the rest 15% of AOX activity [18]. On the other hand, MutS strain does not have the aox1 gene and its AOX activity completely relies on the aox2 gene, thus, MutS strain has a much lower AOX activity and methanol consumption ability than those of Mut+ strain [18, 19]. In this study, sorbitol was co-fed with methanol at a fixed feeding ratio (1:4, g/g), as a result, methanol and sorbitol consumption rates were higher for Mut+ as compared to MutS. 3.4 Kinetic models for methanol and sorbitol utilization Methanol/sorbitol co-feeding for P. pastoris is the process with the following features: 1) carbon is supplied by methanol and sorbitol together, and total carbon consumption rate is a constant at a specific time; 2) methanol (primary carbon source) feeding rate should be relatively high for enough inducing intensity; 3) sorbitol (assisted carbon source) feeding rate should be relatively low and sorbitol should not accumulated in broth, or the methanol consumption rate will decrease, which weakens the inducing intensity [6]. Based on the features, Eq. (2) was designed to describe the relationship between methanol and sorbitol utilization. SMeOH and SF,MeOH are the methanol concentrations in broth and the methanol 11
feeding media; SF,Sor is the sorbitol concentration in the sorbitol feeding media; FMeOH is methanol feeding rate; FSor is sorbitol feeding rate, which is equivalent to the sorbitol consumption rate. R*Sor and n are the parameters of the model. RMeOH is the absolute methanol consumption rate without regarding to the effect of sorbitol addition. According to the characteristics shown in Fig. 5C and 5D, RMeOH for MutS and Mut+ strains could be calculated by Eq. (3) and Eq. (4) respectively, where R*MeOH, K, C and Slimit are parameters of the model. In the future work, a number of runs with different methanol and sorbitol feeding rates will be carried out to obtain modeling data. After evaluating all of the parameters, the kinetic models could be used to describe the methanol/sorbitol co-feeding process of P. pastoris for producing different heterologous proteins. n F S dS MeOH Sor F,Sor FMeOH S F,MeOH RMeOH 1 * (2) dt RSor
RMeOH
* RMeOH S MeOH K S MeOH
(3)
C S MeOH Slimit RMeOH 0 S MeOH Slimit
(4)
4. Conclusion Transcriptional levels of genes in carbon metabolism of a MutS strain and a Mut+ strain under the two extreme conditions of “methanol sufficient-oxygen limited (MS-OL)” and “methanol limited-oxygen sufficient (ML-OS)” were compared. The results revealed that, methanol metabolism of the MutS strain was enhanced under “MS-OL”, while the intensity of methanol metabolism of the Mut+ strain under the two conditions were similar. Subsequently, methanol and sorbitol utilizations in the MutS and Mut+ strains were characterized as: methanol utilization rate of the MutS strain gradually decreased when 12
decreasing methanol concentration; methanol utilization rate of the Mut+ strain unchanged unless methanol concentration decreased to a low-limit. Finally, the above characteristics were described with the developed kinetic models. Acknowledgments The authors thank the financial supports from Natural Science Foundation of China (#21606106), Natural Science Foundation of Jiangsu Province (#BK20150127, #BK20160162), the 111 Project (#111-2-06) of China, the Fundamental Research Funds for the Central Universities (JUSRP51632A, JUSRP11536) and Industry-Education-Research Cooperation Project of Jiangsu Province (BY2016022-15). Reference [1]
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15
Appendix 1 DEGs selected between MS-OL and ML-OS concentration in MutS P. pastoris DEGs selected in methanol metabolism Gene
Gene function
Regulate
Log2Ratio
AOX2
Alcohol oxidase2
UP
0.97±0.17
FLD
Formaldehyde dehydrogenase
UP
1.73±0.71
DAS
Dihydroxyacetone synthase
UP
1.29±0.29
F16P
Fructose-1,6-bisphosphatase
UP
0.89±0.35
RPIA
Ribose-5-phosphate isomerase
UP
0.78±0.05
TAL1
Transaldolase
UP
0.88±0.12
DEGs selected peroxisome biosynthesis metabolism Gene
Gene function
Regulate
Log2Ratio
PEX13
Peroxin13
UP
0.88±0.26
PMP34
Peroxisomal membrane protein34
UP
0.75±0.04
PEX2
Peroxin2
UP
0.72±0.17
PEX3
Peroxin3
UP
0.74±0.08
AGX1
Alanine-glyoxylate aminotransferase
UP
0.80±0.10
PEX5
Peroxin5
UP
0.75±0.16
CAT
Catalase
UP
0.82±0.07
DEGs selected related to respiratory chain Gene
Gene function
Regulate
Log2Ratio
ComplexⅣ5b
Cytochrome c oxidase subunit 5b
UP
0.79±0.18
ComplexⅣ6b
Cytochrome c oxidase subunit 6b
UP
0.85±0.22
ATP synthase
Mitochondrial ATP synthase
UP
0.98±0.27
DEGs related to ribosomal proteins biosynthesis metabolism Gene
Gene function
Regulate
RS6
40S ribosomal protein S6
UP
0.94±0.35
RS27B
40S ribosomal protein S27-B
UP
0.73±0.21
RS21_
40S ribosomal protein S21
UP
0.82±0.17
16
Log2Ratio
RLA0
60S ribosomal protein
UP
0.71±0.08
RL15
60S ribosomal protein L15
UP
1.02±0.32
RS26B
40S ribosomal protein S26-B
UP
0.98±0.11
RL22A
60S ribosomal protein L22-A
UP
1.15±0.17
RS12
40S ribosomal protein S12
UP
0.87±0.22
RS10A
40S ribosomal protein S10-A
UP
1.12±0.09
RS13
40S ribosomal protein S13
UP
0.93±0.20
RS22
40S ribosomal protein S22
UP
0.91±0.06
RS4B
40S ribosomal protein S4-B
UP
0.66±0.13
RL30
60S ribosomal protein L30
UP
0.89±0.31
RL17
60SribosomalproteinL17
UP
0.74±0.09
RS7B_
40S ribosomal protein S7-B
UP
0.96±0.19
RL1B
60S ribosomal protein L1-B
UP
0.92±0.40
RS3A
40S ribosomal proteinS1
UP
0.91±0.14
RS11B
40S ribosomal proteinS11-B
UP
0.89±0.23
RL6B
60S ribosomal protein L6-B
UP
0.73±0.12
RL44
60S ribosomal protein L44
UP
1.02±0.39
DEGs selected between MS-OL and ML-OS concentration in Mut+ P. pastoris DEGs selected in TCA circle Gene
Gene fuction
Regulate
Log2Ratio
PCKA
Phosphoenolpyruvate carboxykinase
DOWN
-1.71±0.11
ACS1
Acetyl-coenzyme A synthetase 1
DOWN
-2.41±0.28
OGDC
2-oxoglutarate dehydrogenase E1
DOWN
-0.78±0.13
IDH2
componentdehydrogenase 2Isocitrate
DOWN
-1.8±0.32
SUCA
oxoglutarate E1 Succinyl-CoAdehydrogenase synthetase
DOWN
-0.63±0.09
CISY
Citrate synthase,
DOWN
-2.56±0.47
FUM
Fumarase
DOWN
-0.87±0.04
17
SDHA
Succinate dehydrogenase
DOWN
-0.94±0.18
ACON
Aconitase
DOWN
-2.17±0.75
DHDL
Dihydrolipoamide dehydrogenase
DOWN
-0.72±0.15
MDHC
Malate dehydrogenase
DOWN
-1.66±0.34
DOWN
-0.76±0.22
DOWN
-1.03±0.37
DOWN
-1.32±0.41
DEGs related to respiratory chain ComplexⅡ
Succinate dehydrogenase cytochrome b560 subunit
ComplexⅡ
Succinate dehydrogenase cytochrome b small subunit
ComplexⅡ
Succinate dehydrogenase flavoprotein subunit
DEGs select in amino acids biosynthesis Genes related to Val, Leu and Ile biosynthesis EC
Threonine dehydratase
UP
0.62±0.13
ILVB
Acetolactate synthase
UP
0.77±0.21
HADH
Dihydroxyacid dehydratase
UP
0.62±0.06
BCA1
Branched-chain-amino-acid
UP
0.65±0.16
UP
0.67±0.07
aminotransferase Genes related to Phe, Tyr and Try biosynthesis AROG
3-deoxy-D-arabino-heptulosonate-7phosphate (DAHP) synthase
CHMU
Chorismate mutase
UP
0.82±0.18
AATC
aspartate aminotransferase
UP
1.32±0.39
HIS8
Histidinol-phosphate aminotransferase
UP
0.88±0.12
TRP
Tryptophan synthase
UP
0.64±0.08
Genes related to Ser, Gly and Cys biosynthesis SERA
D-3-phosphoglycerate dehydrogenase
UP
0.78±0.06
CBS
Cystathionine beta-synthase
UP
0.82±0.11
DEGs selected in fatty acid degradation metabolism Gene
Gene fuction
regulate
Log2Ratio
LCF1
Long chain fatty acyl-CoA synthetase1
DOWN
-1.59±0.43
18
PXA2
Peroxisomal long-chain fatty acid import protein 1
DOWN
-2.95±0.66
LCF2
Long chain fatty acyl-CoA synthetase 2
DOWN
-3.27±0.47
PXA1
Peroxisomal long-chain fatty acid import protein 2
DOWN
-3.16±0.77
THIK
3-ketoacyl-CoA thiolase
DOWN
-2.43±0.73
CACP
O-acetyltransferase
DOWN
-2.78±0.49
PXMP4
Peroxisomal membrane protein 4
DOWN
-1.55±0.67
SODM
Superoxide dismutase [Mn]
DOWN
-0.78±0.28
ACOX
Acyl-coenzyme A oxidase
DOWN
-3.22±0.84
PMP20
peroxiredoxin pmp20
DOWN
-0.90±0.13
SPS19
Peroxisomal 2,4-dienoyl-CoA reductase
DOWN
-1.33±0.34
DEGs in ubiquitin-proteasome system Gene
Gene function
Regulate
RPN13
26S proteasome regulatory subunit RPN13
UP
0.63±0.21
PSB3
Proteasome subunit beta type-3
UP
0.80±0.10
RPN11
26S proteasome regulatory subunit RPN11
UP
0.70±0.29
PRS6B
26S protease regulatory subunit 6B
UP
0.65±0.12
RPN2
26S proteasome regulatory subunit RPN2
UP
0.77±0.21
PSB5
Proteasome subunit beta type-5
UP
0.83±0.11
CDC48
ATPase in ER
UP
0.73±0.17
PDI
Protein disulfide-isomerase
UP
1.17±0.25
OSTB
Dolichyl-diphosphooligosaccharide--protein
UP
0.64±0.08
PNG1
Peptide-N(4)- asparagine amidase
UP
0.59±0.14
Unknown
Ubiquitin ligase complex domain-containing proteins
UP
0.60±0.09
SC61B
Protein transport protein SEC61 subunit beta
UP
0.67±0.10
SC61G
Protein transport protein SEC61 subunit gamma
UP
1.08±0.37
19
Log2ratio
Figure captions Fig. 1 Variations of concentrations of DO, methanol, sorbitol, cell and formate in cell for the MutS and Mut+ strains under the MS-OL and the ML-OS conditions. A, C and E: the results for the production of pIFN-α by the MutS strain; B, D and F: the results for the production of HSA-GCSFm by the Mut+ strain. Filled squares: methanol concentrations under MS-OL condition; open squares: methanol concentrations under ML-OS condition; filled triangles: sorbitol concentrations under MS-OL condition; open triangles: sorbitol concentrations under ML-OS condition; filled circles: cell concentrations under MS-OL condition; open circles: cell concentrations under ML-OS condition; filled diamonds: formate concentrations in cell under MS-OL condition; open diamonds: formate concentrations in cell under ML-OS condition; solid line: DO under MS-OL condition; dash line: DO under ML-OS condition. Fig. 2 Induction performances of the MutS and Mut+ strains under MS-OL and ML-OS conditions. A: variation of pIFN-α concentration in induction phase; B: variation of HSA-GCSFm concentration in induction phase; C: result of SDS-PAGE for the pIFN-α production process; MutS strain; D: result of SDS-PAGE for the HSA-GCSFm production process. Filled circles: target protein concentrations under the MS-OL condition; open circles: target protein concentrations under the ML-OS condition. Fig. 3 Comparison of the expression levels of the key genes in carbon (methanol and sorbitol) metabolism. a: MutS; b: Mut+. Fig. 4 Average methanol and sorbitol consumption rates of the MutS and Mut+ strains under MS-OL and ML-OS conditions. A: result for the MutS strain; B: result for the Mut+ strain.
20
Fig. 5 The MutS and Mut+ strains’ characteristics of utilizing methanol. A and B showed the variations of methanol concentrations in the period when methanol was gradually running out. C and D showed the variations of methanol consumption rates versus methanol concentrations. A and C: result for the MutS strain; B and D: result for the Mut+ strain. Solid lines: methanol concentrations; black dash lines: DO; red dash lines: limit levels of methanol concentration; open circles: methanol consumption rates.
21
6 40 4 2
0
0 0
80
10 8
60
6 40 4 20
2 0
0
10 20 30 40 50 60 70
0
10 20 30 40 50 60 70
Fermentation time after induction (h)
Fermentation time after induction (h)
20
20
Sorbitol conc. (g/L)
C
15 10 5
D
15 10 5 0
0 10 20 30 40 50 60 70
0
Fermentation time after induction (h) 0.2
120 100 80
0.1
60 40 20 0.0
0 0
10 20 30 40 50 60 70
140
Cell conc. (g/L)
E
Formate conc. (g/g-DCW)
140
10 20 30 40 50 60 70
Fermentation time after induction (h) F
120 100 80
0.1
60 40 20 0.0
0 0
Fermentation time after induction (h)
0.2
10 20 30 40 50 60 70
Fermentation time after induction (h)
Fig.1
22
Formate conc. (g/g-DCW)
0
Cell conc. (g/L)
B
Methanol conc. (g/L)
8
60
20
Sorbitol conc. (g/L)
10
DO (%)
A
Methanol conc. (g/L)
DO (%)
80
0.8
A
HAS-GCSF conc. (g/L)
1.5 1.0
m
pIFN- conc. (g/L)
2.0
0.5 0.0
B
0.6 0.4 0.2 0.0
0 10 20 30 40 50 60 70 Fermentation time after induction (h) C
MS-OL
0 10 20 30 40 50 60 70 Fermentation time after induction (h)
ML-OS
ML-OS
D
M 8h 32h 56h 70h 8h 32h 56h 70h
MS-OL
M 8h 32h 56h 70h 8h 32h 56h 70h 150KD
66KD 45KD 35KD 27KD 20KD 14.4KD 9.5KD 6.5KD
100KD 70KD 50KD
HSA-GCSFm 85KD
35KD
pIFN-α 25KD 16KD 20KD 15KD
Fig.2
23
Sor SDH F6P a:ND/b:ND
O2
CH3OH
GAP
Pathway B CH3OH
PYR
Pathway C
O2
Fatty acid AcCoATHIK a:ND/b:-5.38
H2O2
AOX a:0.97/b:ND HCHO
CISY OAA CIT a:ND/b:-2.55 MDHC ACON a:ND/b:-1.65 a:ND/b:-2.17 MAL ICIT FUM a:ND/b:-0.87
TCA
FUM SDHA a:ND/b:-0.94 SUC
HCHO
TAL DHA GAP a:0.97/b:ND R5P Peroxisome
IDH a:ND/b:-1.79 α-KG OGDC a:ND/b:-0.78 Suc-COA
DHA
ATP ADP DHAP GAP
R5P F16P a:0.89/b:ND FBP F6P
Pathway A Targeted Protein
Functional cell
Pi
NADH
FAD
ComplexⅡ a: ND/b: -1.32
CO2
Xy5P
NAD+ ADP ATP synthase a: 0.98/b: ND ATP CoQ ComplexⅢ
NADH 2eComplexⅠ FADH2 2e-
CO2
DAS a:1.29/b:ND
SUCA a:ND/b:-0.63
FADH2
FLD a: 1.73/b:ND FDH HCOOH GS-CH2OH + NAD NAD+ NADH NADH GSH
ComplexⅣ - a: 0.85/ND Cytochrome c 2e 1/2O2+2H+ ATP synthase ATP synthase ADPa: 0.98/b: ND ATP ADP a: 0.98/b: NDATP
Fig. 3
24
H2O
A
10
Methanol Sorbitol
8 6 4 2 0
12
rMeOH, ave, rSor, ave (g/L/h)
rMeOH, ave, rSor, ave (g/L/h)
12
B
10
Methanol Sorbitol
8 6 4 2 0
MS-OL
ML-OS
MS-OL
Fig. 4
25
ML-OS
80
0.6
60
0.3
40
2.0
0.5
1.0 1.5 Time (h)
2.0
4
20 2.5
80
2
60
1
40
5
0.5
1.0 1.5 Time (h)
2.0
20 2.5
D
4
rMeOH (g/L/h)
1.5 1.0 0.5 0.0 0.0
3
0 0.0
C
100
methanol addition
B
3 2 1 0
0.3 0.6 0.9 1.2 Methanol concentration (g/L)
0
Fig. 5
26
1 2 3 4 Methanol concentration (g/L)
DO (%)
0.9
0.0 0.0
rMeOH (g/L/h)
100
A
DO (%) Methanol conc. (g/L)
Methanol conc. (g/L)
1.2