Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose

Abstract

Modification of the Schizosaccharomyces pombe genome is often laborious, time consuming due to the lower efficiency of homologous recombination. Here, we constructed metabolically engineered S. pombe strains using a CRISPR-Cas9 system and also demonstrated D-lactic acid (D-LA) production from glucose and cellobiose. Genes encoding two separate pyruvate decarboxylases (PDCs), an L-lactic acid dehydrogenase (L-LDH), and a minor alcohol dehydrogenase (SPBC337.11) were disrupted, thereby attenuating ethanol production. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced genes encoding bacterial acetylating acetaldehyde dehydrogenase enzymes (Escherichia coli MhpF and EutE). D-LA production by the resulting strain was achieved by expressing a Lactobacillus plantarum gene encoding D-lactate dehydrogenase. The engineered strain efficiently consumed glucose and produced D-LA at 25.2 g/L from 35.5 g/L of consumed glucose with a yield of 0.71 g D-LA / g glucose. We further modified this strain by expressing beta-glucosidase by cell surface display; the resulting strain produced D-LA at 24.4 g/L from 30 g/L of cellobiose in minimal medium, with a yield of 0.68 g D-LA / g glucose. To our knowledge, this study represents the first report of a S. pombe strain that was metabolically engineered using a CRISPR-Cas9 system, and demonstrates the possibility of engineering S. pombe for the production of value-added chemicals.

Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe

“…The PCR products encoding acc , acsSE* , atd1 , and ptk1 were cloned (separately) into pDUAL-FFH-31(RIKEN BRC); the PCR product encoding mcr - c * was cloned into pDUAL-FFH-61(RIKEN BRC). The PCR product encoding mcr - n was cloned between the Nco I and Xho I sites of pDUAL-hsp-SPBC359.04c_BGL [ 17 ]. The resulting plasmid carries mcr - N under the control of the hsp promoter [ 17 ].…”

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…Proof-of-concept: CRISPR for integration of lactic acid producing genes in S. pombe [77] CRISPR Solanum lycopersicum γ-aminobutyric acid (GABA) Proof-of-concept: CRISPR for genome engineering in cyanobacteria [60] employing gRNA libraries. Due to the short length of gRNAs (~ 100nt), accurate predictability, and easy cloning approaches, genome-wide gRNA libraries have been successfully designed to knockout and regulate genes throughout the entire genome [31].…”

Methods of yeast genome editing

“…Так, при помощи разработанного набора инструментов для выполнения метаболической инженерии Schizosaccharomyces pombe с использованием CRISPR/Cas был выделен штамм-продуцент молочной кислоты (Ozaki et al, 2017). Для этого в геноме были но-каутированы шесть генов, встроены три гена и проведена оптимизация их экспрессии.…”

“…Итоговый штамм позволил получить молочную кислоту в концентрации 25 г/л с вы-ходом 0.71 г/г. Дальнейшая оптимизация может увеличить выход конечного продукта до значения, достаточного для организации технологического процесса (Ozaki et al, 2017). Изменения, которые были внесены в ходе выпол-нения работы, представлены на рис.…”

“…Направляющая гидДНК для белков Аргонавт значительно проще в сравнении с гидРНК для Cas9. Размер известных белков Аргонавт со- (Ozaki et al, 2017).…”