Biology subjects

Henard, C. A.

Publications and source records attributed to Henard, C. A..

3 recordsLinked to original sources

bioRxiv2026

Heterologous expression of lyngbyatoxin biosynthetic genes in Aspergillus oryzae reveals transcriptional barriers but enables LtxC-mediated biotransformation

Cyanobacterial natural products are a rich source of bioactive compounds, yet their heterologous production remains challenging. This study investigates the feasibility of expressing the lyngbyatoxin A (LTXA) biosynthetic gene cluster in a fungal host. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed using LC- MS, and transcriptional analysis was performed by RT-PCR. Codon-optimized constructs and precursor feeding experiments were employed to evaluate pathway functionality. No production of LTXA or pathway intermediates was detected upon co-expression of ltxA-C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis revealed truncation of the ltxA transcript, suggesting incompatibility with fungal transcriptional or splicing machinery. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. These results highlight transcriptional limitations as a key barrier to heterologous expression of cyanobacterial NRPS pathways in fungal hosts, while demonstrating that downstream tailoring enzymes can remain functional. This work provides insights for future engineering of fungal platforms for cyanobacterial natural product biosynthesis.

synthetic biology↗

bioRxiv2025

CRISPRi-based functional genomic screening identifies genes essential for CH4-dependent growth in the methanotroph Methylococcus capsulatus Bath.

Aerobic methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH4) and have potential to mitigate the climate-active gas. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a CRISPR interference (CRISPRi) system to enable functional genomic screening in methanotrophic bacteria. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph, Methylococcus capsulatus, consisting of [~]45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH4 identified 233 genes whose transcription repression resulted in a fitness defect and repression of 13 genes associated with a fitness advantage. Enrichment analysis of the 233 putative essential genes linked many of the encoded proteins with critical cellular processes like ribosome biosynthesis, translation, transcription, and other central biosynthetic metabolism, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs. The CRISPRi screen uncovered that 50 ppb Co2+ in methanotroph Nitrate Mineral Salts medium is growth inhibitory and decreasing Co2+ to < 10 ppb improved biomass productivity from CH4. Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify novel genetic determinants of methanotrophy and isolation of improved methanotroph biocatalysts.

microbiology↗

bioRxiv2022

Leishmania amazonensis infection induces PD-L1 expression on dendritic cells in an mTor-dependent manner and impairs Th1 responses in vitro and in vivo

Leishmania amazonensis is one of the etiological agents of diffuse cutaneous leishmaniasis in South America. In murine models of this infection, dysregulated expansion of effector T cells or exhaustion of Th1 responses are known to be related to pathogenesis, while the induction of regulatory T cells (Tregs) promotes lesion resolution. Recent research has identified several important co-stimulator/receptor pairs, including PD-1/PD-L1, for modulating Th1 responses and Treg induction. In this study, we examined the roles of these molecules in L. amazonensis-infected C57BL/6 mice. We found a significant and selective increase in the expression of PD-1 and PD-L1 (20-fold and 5-fold, respectively), in infected footpad tissues, which correlated with an increased percentage of PD-L1+CD11c+ dendritic cells (DCs) and PD-1+CD4+ T cells in the draining lymph nodes. To evaluate the mechanism of parasite-induced PD-L1 expression, we infected bone marrow-derived DCs (BMDCs) with promastigotes and amastigotes in vitro in the presence of small molecular inhibitors of critical signaling pathways. While L. amazonensis infection decreased PD-L2 expression on the surface of BMDCs, infection-mediated PD-L1 up-regulation was consistently detected, which was dependent on mTOR and partially dependent on STAT3, PI3K, and MAPK. Infected BMDCs also significantly inhibited the expansion of Th1 cells, but were more competent in inducing CD25+FoxP3+ Tregs in vitro than the non-infected BMDCs; such effects were PD-L1-dependent, as BMDCs from PD-L1-/- mice failed to do the same. In vivo experiments revealed that infected PD-L1-/- mouse tissues showed increased Th1 responses and IFN-{gamma} production, as well as reduced lesion sizes and parasite loads, without affecting Tregs cell expansion. Together, these results support a protective role for PD-1/PD-L1 signaling in regulating local immune responses during L. amazonensis infection. This study provides new insights on immune regulation in New World cutaneous leishmaniasis.

immunology↗